While a great deal has been learned about the neuropharmacology of addiction in recent years, we still have no biological treatments that can prevent or reverse the adaptations in brain function that highjack the mechanisms involved in normal motivated behavior. Brain regions such as the striatum are key targets for developing novel treatments for addiction, but it is important that these treatments do not disrupt normal information processing involved in routine motivated behaviors. This has been a particular problem with dopaminergic drugs, but other neurotransmitters such as serotonin are also centrally involved in the biology of addiction. One potential target is the 5-HT6 serotonin receptor, which is heavily expressed in striatum and affects procedural learning that is involved in drug reward and habit formation. Additionally, the 5-HT6 receptor is the only serotonin receptor localized to the primary neuronal cilium, a cellular organelle present on most neurons that has a critical role in brain development and normal cognitive function that has not yet been thoroughly characterized. Since cilia contain a number of cellular signaling systems that modulate gene transcription and plasticity without interfering with synaptic function directly, thi suggests that exploring the significance of 5-HT6 localization in cilia is a crucial next step. Ciliary localization is thought to be governed by a discrete consensus sequence in the third intracellular loop of GPCRs including 5-HT6 and a small set of other proteins. This R21 project has two aims. The first aim will focus on making site direct mutations in 5-HT6 receptors that will prevent them from localizing to cilia without perturbing their molecular functions otherwise; we will then investigate the signaling consequences of activating 5-HT6 receptors that are or are not localized in cilia. We will perform most of these experiments in primary cultured striatal neurons prepared from 5-HT6 knockout mice, providing a strong basis for reaching conclusions about their function. We will examine the effects of 5-HT6 receptors on cilia and dendritic morphology, cyclic AMP production, DARPP32 hosphorylation, and gene expression using PCR arrays focusing on key pathways predicted to be sensitive to 5-HT6 and cilia function. In the second aim we will test whether ciliary localization of 5-HT6R receptors is critical to the well- established behavioral effects of expressing 5-HT6 receptors in dorsomedial striatum using instrumental learning as the key behavioral outcome. We will perform these experiments both in rats (where the effect is well established) and in wild-type and 5-HT6 knockout mice, where the effects of normal or mutant 5-HT6 receptors can be studied in the presence and absence of endogenous 5-HT6 receptors. These experiments will create a platform for determining whether targeting cell signaling mechanisms in the cilia will offer new opportunities for developing therapeutics for addiction and other compulsive disorders.